Energy Stress Causes Chaperones to Assemble Into Cytoplasmic Complexes

The Texas Medical Center Library DigitalCommons@TMC

The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences

8-2014

ENERGY STRESS CAUSES CHAPERONES TO ASSEMBLE INTO CYTOPLASMIC COMPLEXES

Kimberly J. Cope

Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations

Part of the Cell Biology Commons, Laboratory and Basic Science Research Commons, Microbiology Commons, and the Molecular Biology Commons

Recommended Citation Cope, Kimberly J., "ENERGY STRESS CAUSES CHAPERONES TO ASSEMBLE INTO CYTOPLASMIC COMPLEXES" (2014). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 510. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/510

This Thesis (MS) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. ENERGY STRESS CAUSES CHAPERONES TO ASSEMBLE INTO CYTOPLASMIC COMPLEXES Formatted: No widow/orphan control

Formatted: Different first page header by

Kimberly Jaclyn Cope, B.S.

APPROVED:

______Advisory Professor, Kevin A. Morano, Ph.D.

______Michael Lorenz, Ph.D.

______William Margolin, Ph.D.

______Ambro van Hoof, Ph.D.

______Eric J. Wagner, Ph.D.

APPROVED:

______Dean, The University of Texas Graduate School of Biomedical Sciences at Houston

ENERGY STRESS CAUSES CHAPERONES TO ASSEMBLE INTO CYTOPLASMIC COMPLEXES

THESIS

Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment

of the Requirements

for the Degree of

MASTER OF SCIENCE

Kimberly Jaclyn Cope, B.S. Houston, Texas

August 2014

Formatted: Centered

Formatted: No widow/orphan control

Formatted: Centered iii

Formatted: No widow/orphan control

Formatted: Centered iv

Dedication Formatted: Left, Space After: 8 pt, Line spacing: Multiple 1.08 li, No widow/orphan control, Adjust space between Latin and Asian text, Adjust space between Asian text and numbers Formatted: No widow/orphan control

Formatted: Centered v

Acknowledgments Formatted: Space After: 0 pt, Line First and foremost, I would like to thank my advisor Dr. Kevin Morano for allowing me the spacing: single, No widow/orphan control Formatted: Indent: First line: 36 pt, Line opportunity to work in his laboratory and guiding me through both the triumphs and failures of spacing: Double, No widow/orphan control graduate school in the sciences. I would like to thank all members of my committee, past and Formatted: Font: Not Bold present, including Dr. Michael Lorenz, Dr. William Margolin, Dr. Ambro van Hoof, Dr. Eric Wagner, Formatted: Font: Not Bold

Dr. Danielle Garsin, Dr. Theresa Koehler, and Dr. Steve Norris. I’d like to thank past and present members of the Morano laboratory for their support: Dr. Patrick Gibney, Dr. Hugo Tapia, Dr. Jacob

Verghese, Dr. Yanyu Wang, Dr. Jennifer Abrams, Veronica Garcia, Sara Peffer, Amy Ford, and Julie

Heffler. Finally I would like to thank all my friends and family for helping me be the person I am today. Formatted: Font: Not Bold

Formatted: Centered vi

ENERGY STRESS CAUSES CHAPERONES TO ASSEMBLE INTO CYTOPLASMIC COMPLEXES Formatted: No widow/orphan control

Kimberly Jaclyn Cope, B.S.

Advisory Professor: Kevin. A. Morano, Ph.D.

The majority of proteins require molecular chaperones to assist their folding into tertiary Formatted: Justified, Line spacing: Double, No widow/orphan control and quaternary structures. Certain stresses can compromise the weak hydrophobic forces responsible for these structures and lead to protein unfolding, misfolding, and aggregation.

Aggregates of proteins are hallmarks of devastating diseases such as Alzheimer’s, Parkinson’s, and

Huntington’s diseases. Fortunately, bacteria, plants, and fungi have a potent disaggregase, named

Hsp104 in Saccharomyces cerevisiae .

Recently, heat-induced aggregates, termed Q-bodies, were found to contain three molecular chaperones: Hsp70, Hsp104, and Hsp42. Their coalescence from small puncta into larger inclusions requires Hsp104. During glucose deprivation, a stress that isn’t known to cause unfolding of proteins, I have observed Hsp104, Hsp26, and Hsp42 but not Ssa1 form cytoplasmic foci that I have termed chaperone bodies (CBs). As the Q-bodies are hypothesized to be involved in protein refolding or degradation, the chaperone bodies may also play a crucial role in protein quality control. The overall goal of my project was to characterize these novel bodies in terms of composition and dynamics.

The different chaperones coalesce into aggregates at different frequencies in response to Formatted: No widow/orphan control, Don't adjust space between Latin and starvation, however a model thermally labile protein (firefly luciferase) does not, suggesting that Asian text, Don't adjust space between Asian text and numbers misfolded proteins are not responsible for the recruitment of the chaperones. One hypothesis is that energy depletion is hampering the ability of certain proteins to be anchored into the membrane post-translationally, exposing hydrophobic residues prone to aggregation. By performing the glucose Formatted: Centered vii deprivation in the presence of cycloheximide, a translation inhibitor, I observed that, indeed, newly synthesized proteins must be present for the formation of CBs.

To determine whether CBs colocalize and share a purpose with other known foci, I Formatted: Indent: First line: 36 pt, Line spacing: Double, No widow/orphan compared the localization of the chaperones to representative markers in live cells. After acute control, Adjust space between Latin and Asian text, Adjust space between Asian text glucose starvation, Hsp104 partially colocalizes with proteasome storage granules and fully overlaps and numbers with stationary phase granules. I observed that Hsp104’s localization patterns were partially dependent on the nature of the fluorescent protein attached. Fusion to yEmRFP caused Hsp104 to remain mostly diffuse during starvation, while fusion to CFP caused it to localize to structures that resemble the septin ring and Gln1 filaments. CFP also caused an apparent cytokinesis defect. I hypothesize that the subtle differences between the fluorescent proteins may be interfering with

Hsp104 assembly and function. Overall, based on my data, CBs appear to be a novel response to nutrient stress, warranting further investigation.

Formatted: Indent: First line: 36 pt, No widow/orphan control

Formatted: Centered viii

Text Formatted: Indent: First line: 36 pt, Line spacing: Double, No widow/orphan control, Adjust space between Latin and Asian text, Adjust space between Asian text and numbers Formatted: Line spacing: Double, No widow/orphan control Formatted: No widow/orphan control

Formatted: Centered ix

Table of Contents Formatted: Space After: 8 pt, Line spacing: Multiple 1.08 li, No widow/orphan control, Adjust space Approval Sheet ...... i between Latin and Asian text, Adjust space Title Page ...... ii between Asian text and numbers Formatted: No widow/orphan control Acknowledgements ...... iii Abstract ...... iv Table of contents ...... vii List of Figures ...... viii List of Tables ...... ix Abbreviations ...... x Introduction ...... 1 Methods ...... 10 Results ...... 16 Discussion ...... 28 References ...... 38 Vita ...... 41

Formatted: Centered x

List of Figures Formatted: Justified, Space After: 0 pt, Line spacing: single, No widow/orphan control, Don't adjust space between Latin Figure 1: Unfolding of mature proteins ...... 3 and Asian text, Don't adjust space between Asian text and numbers Figure 2: Chaperone involvement in Q-body formation and dynamics ...... 5 Formatted: No widow/orphan control Figure 3: Disaggregation by Hsp104 ...... 6 Formatted: Font: Not Bold Figure 4: Structure of Hsp104 ...... 8 Formatted: Line spacing: 1.5 lines, No Figure 5: Hsp26 dynamics ...... 10 widow/orphan control, Tab stops: 450 pt, Right,Leader: … + Not at 387 pt Figure 6: Heat- and starvation-induced bodies ...... 22 Formatted: Font: Not Bold Figure 7: Additional stresses ...... 24 Formatted: Font: Not Bold Figure 8: Foci formation in the presence of cycloheximide ...... 28 Formatted: Line spacing: 1.5 lines, No widow/orphan control, Tab stops: Not at Figure 9: Colocalization with additional granule markers ...... 29 418.5 pt + 423 pt Figure 10: Fluorescent-protein-dependent localization of Hsp104 ...... 31 Formatted: Font: Not Bold Formatted: Font: Not Bold Formatted: Font: Not Bold Formatted: Justified, Space After: 0 pt, Line spacing: single, No widow/orphan control, Don't adjust space between Latin and Asian text, Don't adjust space between Asian text and numbers

Formatted: Centered xi

List of Tables Formatted: No widow/orphan control

Table 1: Genomic chaperone fusions ...... 12 Formatted: Line spacing: 1.5 lines, No Table 2: Plasmid chaperone fusions ...... 13 widow/orphan control, Tab stops: 450 pt, Right,Leader: … + Not at 423 pt Table 3: C-terminal tagging plasmids ...... 15 Table 4: Additional plasmids ...... 16

Formatted: Font: Not Bold

Formatted: No widow/orphan control

Formatted: Centered xii

Abbreviations

CFP: cyan fluorescent protein

GFP: green fluorescent protein

EGFP: enhanced green fluorescent protein yEmRFP: yeast enhanced red fluorescent protein

Formatted: Centered xiii

Formatted: No widow/orphan control

Formatted: Centered 1

I. Introduction

Overview

In the following thesis, I explore a new phenomenon of heat shock protein 104 (Hsp104) and the small heat shock chaperones Hsp26 and Hsp42: coalescence into cytoplasmic bodies in response to acute glucose deprivation. I define preliminarily refer to these complexes as chaperone bodies

(CBs). Further, I explore the formation and composition of these CBs and describe future experiments that can be undertaken to examine the role of these complexes in protecting or sequestering particular proteins during nutritional stress. The results indicate that CBs are a novel mechanism for dealing with energy stress and additional study may provide insight into general protein quality control pathways. Formatted: Font color: Red

Prote ostasis Protein homeostasis

In both prokaryotic and eukaryotic cells, the majority of proteins require molecular chaperones to assist their folding from an unstructured peptide into a functional conformation defined as the “native state ” [1]. This system is highly conserved across all kingdoms, with much of our knowledge on the subject coming from the model eukaryote , S. cerevisiae [2]. Molecular chaperones are not components of the final protein and do not contain any st ructural eric information, instead they sim ply bind and stabilize unstable intermediates through hydrophobic interactions [3]. In fact, all folding information is contained within the amino acid sequence of the translated protein and single-domain proteins can often fold spontaneously from denatured poly peptides in vitro [4]. However, the cellular environment differs from the test tube, making chaperones necessary in vivo . First, the local concentration of nascent polypeptide chains is very high due in part to the fact that ribosomes are organized into polysomes with more than one translato ribosome per transcript [3]. Upon exit from the ribosome, the poly peptides assemble into Formatted: Centered 2 alpha helices and beta sheets with loose hydrophobic interactions [3]. The se loose interactions between the se secondary structures allow them to easily unfold and aggregate with similar residues of neighboring nascent chains [3]. Without chaperones, the nascent chains would become tangled with themselves and each other. Second, the cytosol is very crowded with other macromolecules

(for example, E. coli contains around 340 g of macromolecules per liter of cytosol), and this high concentration can increase association constants several orders of magnitude compared to the dilute environment a dilute within a test tube [3]. Without molecular chaperones, the nascent chains can aggregate with nearby proteins, rendering them non-functional. Third, proteins are translated vectorially but but the first two hydrophobic residues that emerge will are not necessarily intended to bind one another be bound in the final conformation . Without molecular chaperones to Comment [KAM1]: Awkward sentence recognize and bind these residues until an entire domain of the protein is ready to fold, erroneous interactions would could form .occur.

Once a protein has reached its native state, the role of the molecular chaperone is not over conlcuded concluded (Figure 1) . Variations in temperature, ionic strength, or redox state as well as the presence of reactive oxygen species, heavy metals, or detergents can compromise the integrity of the hydrophobic bonds, leading to protein unfolding [5]. Until recently, cells were thought to have two separate lines of defense against misfolded proteins. In the first line of defense line , cells would respond by either degrading them such substrates via the proteasome [23] or lysosome [24], or refolding them via the Hsp70/Hsp40/ HNEF sp ch 110 ch aperone machinery [25].

However, if the stress was too extreme or prolonged, the proteolytic and refolding systems were thought to be come overwhelmed and the increasing accumulating number of unfolded proteins would therefore begin to coalesce into amorphous aggregates via their exposed hydrophobic residues [42]. Recent evidence, however, suggests that the proteostasis machinery does not need to be overwhelmed for aggregates to appear; instead, dynamic inclusions rapidly coalesce at the Formatted: Centered 3 beginning of the stress and remain during pr oteasomal degradation [6] . These early aggregates were have been recently termed q-bodies Q-bodies and were discovered using the a thermally -labile

Formatted: Font: Bold Formatted: Indent: First line: 0 pt, No widow/orphan control

Figure 1: Native proteins and nascent chains c an become unfolded and misfolded , respectively, due Formatted: Font: Bold Formatted: Font: Not Bold to various stresses . These nonfunctional conformers can be degraded by the proteasome, refolded Formatted: Font: Not Bold by Hsp70, Hsp40, and Hsp110, or can form into aggregates. Formatted: Font: Bold

Formatted: No widow/orphan control

Formatted: Centered 4 protein Ubc9ts, which denatures above 33°C [6]. At the non-permissive temperature, Ubc9ts forms Formatted: Indent: First line: 0 pt, No widow/orphan control dim puncta throughout the cytosol. Upon proteasome inhibition, these puncta coalesce into 1-3 large inclusion bodies s which resemble the insoluble protein deposit (IPOD), a compartment of ubiquitinated proteins that colocalizes with proteasomes, and the juxtanuclear quality control

(JUNQ) compartment, which contains terminally misfolded proteins, including amyloids (described below). The initial puncta formed differ from the IPOD and JUNQ compartments in that they colocalize with the cortical endoplasmic reticulum (ER) and form independently of the cytoskeleton.

ATP depletion by sodium azide did not affect formation of the q-bodies Q-bodies , but it disrupt sed their movement, coalescence, and clearance due to the fact that molecular chaperones awe re greatly involved in q-body Q-body dynamics (Figure 2) . Both Hsp42 and Hsp104 were shown to colocalize with q-bodies Q-bodies and Hsp104 was shown to be essential for the coalescence of the smaller puncta into larger inclusions [6].

In addition to Besides amorphous aggregates, cells can also contain more structured Formatted: No widow/orphan control aggregates called amyloids. Amyloid-like plaques were first seen in E. coli expressing recombinant proteins: rather than remaining diffuse throughout the cytosol the exogenously-expressed non - endogenous proteins would precipitate into insoluble inclusion bodies [7]. These aggregates are similar to the amyloid aggregates of eukaryotic cells: they interact with chaperones, they contain a single protein species that is partially unfolded and arranged in beta sheets, they are able to nucleate additional bodies, and they are often toxic [8]. Non-toxic amyloids can be found in the

[PSI+] and [URE3] prions of S. cerevisiae in which misfolded proteins are inherited without devastating cytotoxic effects [9]. In fact , these prions may provide a non-genetic but heritable mechanism to supply phenotypic diversity to populations [10].

Fungi, bacteria, and plants contain a highly conserved disaggregase, named Hsp104 in yeast Formatted: Indent: First line: 36 pt, No widow/orphan control [11] which, in conjunction with the Hsp70/Hsp40 machinery, is able to can extract proteins from Formatted: Centered 5 these aggregates and refold them into functional units (Figure 3) [12-14]. In support of the functional

importance of this property, The the presence of Hs p104 increases the chances of a heat -stressed cell’s survival 10,000

Figure 2: Hsp42 is involved in formation of small, Q -body inclusions while Hsp104, Hsp70, and Hsp90 Formatted: Font: Bold are involved in the coalescence of the small inclusion into larger ones. Formatted: No widow/orphan control

Formatted: Centered 6

Figure 3: Hsp104 can dissolve aggregates and, with the help of Hsp70 and its cochaperones, refold Formatted: Font: Bold them into functional proteins. In metazoans, the Hsp70/Hsp40/Hsp110 machinery h as been shown to have its own disaggregase activity.

Formatted: Centered 7 importance of this property, the presence of Hsp104 increases the chances of a heat-stressed cell’s Formatted: Indent: First line: 0 pt, No widow/orphan control survival 10,000 fold [15]. Additionally, Hsp104 is required for induced thermotolerance, which is defined as the ability of cells to survive a short period s of time at a lethal temperature s when previously exposed to a sub-lethal heat shock [16]. Besides re -solubilizing aggregates of unfolded proteins, Hsp104 also plays roles in prion inheritance and remodeling of Spa2, a member of the polarisome complex responsible for keeping damaged proteins from being transferred to daughter cells during cell division [17, 18].

Each monomer of Hsp104 consists of an N-terminal domain, two AAA+ nucleotide-binding domains (NBD1 and NBD2) linked by a coiled-coil middle domain, and a C-terminal domain (Figure

4A) [12]. To become active, the monomers converge into a hexameric structure resembling a three- tiered barrel: the top tier consists of the N-terminal domains, the middle tier consists of the NBD1s, and the bottom tier consists of the NBD2s, with the coiled-coil domains intercalating between the middle and bottom tiers (Figure 4B) [19, 20]. The formation of this six-subunit structure requires the binding of either ADP or ATP to each of the NBD2s [21, 22], while the binding of substrates to the completed hexamer requires the binding of ATP to the NBD1s as well as presentation of substrates to Hsp104 by Hsp70 [23-25]. In addition to assisting recognition of substrates, Hsp70 and Hsp40 may also influence ATP hydrolysis by Hsp104, which is the driving force in disaggregation [26, 27]. The

NBD1s bind ATP, causing a conformational change that allows an alpha-helical loop in each of the

NBD1s containing a conserved tyrosine residue to move towards the N-terminal opening and bind substrate [20]. Hydrolysis of ATP by the NBD1s allows the substrate-bound loops to return to their original position, pulling the substrate into the barrel structure [20]. By an unknown mechanism beta-hairpin loops in the NBD2s also carrying conserved tyrosine residues sense the ATP hydrolysis by the NBD1s, rotate towards the centrally located substrate, and bind the substrate [20]. The

NBD1s release their ADPs and bind new ATPs, resulting in the NBD1s returning to their positions Formatted: Centered 8 near the moving back upward to the N-termini and the

Formatted: Font: Bold Formatted: Centered

Figure 4: The structure of Hsp104 from S. cerevisiae showing the N -terminal domain (pink), nucleotide binding domain 1 (orange), the middle domain (green), and nucleotide binding domain 2

(blue). Panel A shows the monomer while panel B shows the hexamer [28].

Formatted: Indent: First line: 0 pt

Formatted: Centered 9 substrate-bound NBD2s moving downward to the C-termini [20]. Finally, ATP hydrolysis by the Formatted: Indent: First line: 0 pt, No widow/orphan control NBD2s causes the ejection of the substrate through the bottom of the barrel [20].

T Formatted: No widow/orphan control

[28] The two small heat shock proteins, Hsp26 and Hsp42, are energy-independent holdases that Formatted: Left, Indent: First line: 0 pt, Space After: 8 pt, No widow/orphan bind to unfolded proteins and facilitate their future solubilization [29]. During exponential phase , control

Hsp26 exists as a globular 24-mer that dissociates into dimers during stress [2]. Each of the dimers can bind one substrate unit and then these units can form into larger, ordered complexes. Hsp42 is slightly different, beginning as a symmetric assembly of 12- to 16-mers during exponential phase, then organizing into dimers which further organize into hexameric rings once bound to unfolded substrate. Hsp42 binds promiscuously with 30% of non -native proteins, and about 90% overlap exists between the substrates of Hsp42 ’s and Hsp26 ’s substrates [2].

The unfolding and misfolding of proteins is related to many devastating and incurable aging- related human diseases including Alzheimer’s, Huntington’s, Parkinson’s, type II diabetes, and prion diseases [30-33], all of which involve the spontaneous refolding of proteins into non-functional, beta-sheet-rich amyloid forms [34, 35]. While the amyloid form is extremely stable, being resistant to proteases, detergents, chaotropes, and high temperatures [36-38], it may be unable to withstand the disaggregation activity powe r of Hsp104. Even though recent data have shown that metazoan cells appear to use the unrelated chaperone Hsp110 (known mainly as a nucleotide exchange factor for Hsp70s [39]) for the disaggregation of proteins via a currently poorly understood mechanism[40], the following studies involving Hsp104 and amyloid plaques associated with

Alzheimer’s, Parkinson’s and Huntington’s diseases have produced intriguing results pointing to the possibility of the use of Hsp104 to treat human diseases via gene therapy. In an in vitro study,

Hsp104 prevented the de novo formation of Aβ42 amyloids, which are responsible for the neurodegeneration of the cerebral cortex and subcortical regions in Alzheimer’s patients [41], even Formatted: Centered 10 when the Aβ42 monomers were present at a concentration 1000-fold higher than Hsp104 [42]. In a rat model of Parkinson’s, Hsp104 prevented

Figure 5: Hsp26 begins as a 12-16 -mer during expone ntial phase. Upon stress, it dissociates into Formatted: Font: Bold dimers that can each bind one unfolded protein. The dimers assemble into hexameric rings , keeping the unfolded proteins segregated from each other.

Formatted: Centered 11 and reversed the formation of the α-synuclein plaques that would have otherwise destroyed Formatted: Indent: First line: 0 pt, No widow/orphan control dopaminergic neurons in the substantia nigra pars compacta [43, 44]. In a mouse model of

Huntington’s, Hsp104 reduced polyQ aggregation and increased lifespan 20 percent [45]. Since heterologous expression of Hsp104 is highly tolerated in animal systems and the protein is able to work in conjunction with the metazoan Hsp70/Hsp40 chaperones [24, 46-48], the use of Hsp104 in gene therapy is a promising possibility that warrants thorough investigation of each of Hsp104’s cellular activities.

Formatted: No widow/orphan control

Spatial Quality Control

Besides aggregates, eukaryotic cells are known to form regulated assemblies of natively folded proteins in response to various stresses. Some examples of these cytoplasmic complexes include p-bodies P-bodies , stress granules, actin bodies, proteasome storage granules (PSGs), and stationary phase granules (SPGs) [49-52]. In fact, in a genome-wide study, 180 different proteins, the majority involved in metabolism or stress response, transitioned from a diffuse soluble state to a punctate localization upon entry into stationary phase (defined as 48 hours in synthetic medium plus glucose) [53]. The puncta were not targeted for vacuolar or autophagosomal degradation and they did not localize with major organelles [53]. As a disaggregase, Hsp104 may be recruited to one or more of these bodies to aid in their rapid assembly in response to stress and/or disassembly in response to stress cessation .cessation.

The functions of these bodies are not yet known, and the current hypotheses suggest additional possibilities for the presence of Hsp104. First t The various assemblies have been speculated to protect their constituents from degradation during times of stress. For example, the complexes discovered in the genome-wide search for stress-induced bodies were shown to be resistant to autophagy, a program of degradation that S. cerevisiae initiates in response to Formatted: Centered 12 starvation [53, 54]. In the case of actin bodies, F-actin that has coalesced into the assemblies during stationary phase is quickly redistributed into functional actin cables and patches upon nutrient replenishment [50]. This redistribution occurs in the absence of de novo protein synthesis (achieved via addition of cycloheximide), demonstrating that F-actin held within the actin bodies is resistant to degradation [50]. Similarly, upon exit from stationary phase, proteasome subunits are relocalized from proteasome storage granules to the nucleus in the presence of cycloheximide, implying that these assemblies also serve to protect their constituents since damaged proteins would not be readily shuttled into the nucleus [55]. The constituents of stationary phase granules are also transported back to the nucleus in the absence of de novo protein synthesis when the cell resumes Formatted: Font: Italic proliferation, suggesting again a protective role for the granules [52]. Along these same lines,

Hsp104 may be accumulating into bodies in order to remain protected during certain stresses.

Hsp104 functions as a hexamer and requires 6 six molecules of ATP for assembly [19] , so not having to synthesize it anew could save time and energy. Alternatively, Hsp104 may be facilitating the assembly or disassembly of a body intended to protect an additional protein.

A second hypothesis for the function of the various cytoplasmic bodies is that they increase the local concentration of particular proteins. For example, cajal bodies found in metazoan cells accumulate the small nuclear RNAs U4 and U6 in order to more efficiently assemble them into splicesomal snRNPs [56]. Similarly, stress granules may serve to poise translation initiation factors on mRNAs to ready the cell for a speedy return to protein synthesis when conditions become favorable [49]. In the same vein, the assemblies containing Hsp104 may exist to increase the concentration of Hsp104 to effectively disassemble or remodel various aggregates or multi-protein complexes during particular stresses, or they may be built by Hsp104 to increase the concentration of other constituent proteins.

A third hypothesis is that the stress-induced complexes function to sequester certain Formatted: Centered 13 constituents. Stress granules are believed to sequester RACK1 from activating a MAP kinase pathway that would lead to apoptosis [57]. Both p-bodies P-bodies and stress granules may serve as a buffering system to sequester a certain amount of mRNAs from the cytoplasm so that the translation machinery does not become overwhelmed and the cytoplasm crowded with mRNA awaiting translation [58]. Additionally, the p-bodies P-bodies and stress granules may seclude abundant transcripts, giving rarer ones the opportunity to access the ribosome [58]. Stationary phase granules (SPGs) have been hypothesized to sequester the Tup1-Ssn6 co -repressor, which represses 10% of the genes that are observed to be upregulated during stationary phase; thus, the

SPG may be involved in regulating genes that achieve the quiescent state [59, 60]. The CBs, then, may serve to sequester Hsp104 to conserve energy or prevent the disaggregation of certain complexes during stress. It has been suggested that different prion states may provide phenotypic variation that could be beneficial under certain selective pressures, and sequestering Hsp104 from these prions could lead to a fitness advantage [10]. Or Hsp104 may play a role in the dynamics of a body responsible for sequestering other proteins.

Therefore we set out to understand these novel chaperone bodies in terms of composition and dynamics. Which chaperones coalesce into CBs under what stresses and to what degrees? What other proteins are incorporated into CBs: are they misfolded, newly synthesized, and/or components of other known bodies? Answers to these questions will help us uncover the purpose and significance of CBs.

Formatted: Centered 14

II. Methods

Construction of genomic fusions of chaperones to fluorescent proteins: The fluorescent moiety was

PCR amplified from the specified plasmid (See Table 1) using primers engineered to add regions homologous to 30 bp upstream and downstream of the stop codon of the specified chaperone to the 5’ and 3’ ends of the product, respectively. BY4741 cells were grown to exponential phase and incubated in 10 mM Tris + 1mM EDTA + 0.1M LiAc for 20 min at 30°C. The above construct as well as

10-fold salmon sperm DNA (as a carrier) was added to the cells along with 40% polyethylene glycol +

10 mM Tris + 1 mM EDTA + 0.1 M LiAc. Cells were incubated at 30°C for 30 min. 1/10 volumes of

DMSO was added and the cells were heat shocked at 42° for 6 min. Cells were plated on the appropriate selective media (See Table 1). Each strain was verified via Western blot and sequencing by Genewiz.

Media: Cells were grown in synthetic complete media (SC) from Sunrise Science Products supplemented with yeast nitrogenous base (YNB) and 2% glucose. For glucose deprivation, glucose was omitted. 2-deoxy-D-glucose was added to a final concentration of 20 mM. Sodium azide was added to a final concentration of 20 mM. Potassium chloride was added to a final concentration of

1M. Sorbitol was added to a final concentration of 1M. For nitrogen deprivation, YNB lacking ammonium sulfate was used. Cycloheximide was added to a final concentration of 1 μg/mL.

Creation Construction of C-terminal tagging plasmids: For pFA6a-yEmRFP-KanMX6, yEmRFP was PCR amplified off of yEp-GAP-Cherry-GAP-ALG7 (from the Lorenz lab) using primers engineered to add a

PacI and AscI sites to the 5’ and 3’ ends of the product, respectively. Both the PCR product and pFA6a-GFP(S65T)-KanMX6 [61] were digested with the above enzymes and the yEmRFP portion was ligated into pFA6a-GFP(S65T)-KanMX6 in place of GFP. The same procedure was followed for pFA6a- Formatted: Centered 15

CFP-KanMX6, except the fluorescent protein was amplified from off of pRP1768 41 3-Pab1 -CFP [62].

For pFA6a-yEmRFP-His5MX6 and pFA6a-CFP-His5MX6, the backbone plasmid was pFA6a-GFP(S65T)-

His5MX6 [61].

Stress induction procedures: 5 mL overnight cultures were subcultured to 10 mL of 0.06 OD 600 units in triplicate. Cells were incubated at 30°C with aeration for 6 hours to reach 0.9 OD 600 units, centrifuged at 3000 x g for 1.5 min, washed in 1 mL of experimental media, recentrifuged at 3000 x g for 1.5 min, and resuspended in 10 mL of experimental media, and incubated for 15 or 60 minutes

(depending on the experiment) at 30°C with aeration. After incubation, cells were centrifuged for

1.5 min at 3000 x g, decanted to leave around 1 mL of solution with the pellet, resuspended in this solution, transferred to a microcentrifuge tube, centrifuged at 3000 x g for 1.5 min , decanted to leave around 100 μL of solution with the pellet, and then resuspended in this solution. 1.8 μuL were pipetted onto a microscope slide, a coverslip was placed over the droplet and slides viewed immediately on an IX81 motorized inverted microscope equipped with a Hamamatsu ImagEM camera and Slidebook 5.5.4 software from 3I: Intelligent Imaging Innovations. Images were exposed for 2 sec for fluoresescent fluorescent light and 5 msec for DIC.

Formatted: Centered 16

Phenotype of Chaperone with Strain Name Marker Source of Fluorescent Protein Notes Formatted: No widow/orphan control Acute Glucose Deprivation gHsp42-GFP His3MX6 pFA6a-GFP(S65T)-His3MX6 Increased Foci Purchased from Invitrogen Formatted: Bottom: 90 pt, Width: 792 pt, gHsp104-GFP His3MX6 pFA6a-GFP(S65T)-His3MX6 Increased Foci Purchased from Invitrogen Height: 612 pt gHsp26-GFP KanMX6 pFA6a-GFP(S65T)-KanMX6 Increased Foci Created by Kirsten Davis gHsp104-yEGFP Kan pKT127 from EuroSCARF Increased Foci gHsp104-yEmRFP KanMX6 pFA6a-yEmRFP-KanMX6 Stable Number of Foci gCap1-GFP, Hsp104-yEmRFP His3MX6 and KanMX6 pFA6a-GFP(S65T)-His3MX6 and pFA6a-yEmRFP-KanMX6 Stable Number of Foci gScl1-GFP, Hsp104-yEmRFP His3MX6 and KanMX6 pFA6a-GFP(S65T)-His3MX6 and pFA6a-yEmRFP-KanMX6 Stable Number of Foci gHos2-GFP, Hsp104-yEmRFP His3MX6 and KanMX6 pFA6a-GFP(S65T)-His3MX6 and pFA6a-yEmRFP-KanMX6 Stable Number of Foci gHst2-GFP, Hsp104-yEmRFP His3MX6 and KanMX6 pFA6a-GFP(S65T)-His3MX6 and pFA6a-yEmRFP-KanMX6 Stable Number of Foci gHsp104-CFP KanMX6 pFA6a-CFP-KanMX6 Septin gHsp104-yEmCFP His5 pKT210 from EuroSCARF Increased Foci gHsp104-yECitrine Kan pKT140 from EuroSCARF Increased Foci

Table 1: Genomic fusions of chaperones and fluorescent proteins

Formatted: Centered

Phenotype of Chaperone with Name Marker Source of fluorescent protein Acute Glucose Deprivation p413-Hsp26-yEmRFP KanMX6, His3 pFA6a-yEmRFP-KanMX6 Diffuse p416-Hsp104-RFP Ura3 pLM1002 (pRS416-Hsp104-RFP) No Foci p416-Hsp26-ECFP KanMX4, Ura3 pYM30 from EuroSCARF Septin p416-Hsp42-ECFP KanMX4, Ura3 pYM30 from EuroSCARF Septin p416-Hsp104-ECFP KanMX4, Ura3 pYM30 from EuroSCARF Septin p413-Hsp26-yEmCFP His3, His5 pKT210 from EuroSCARF Septin p413-Hsp42-yEmCFP His3, His5 pKT210 from EuroSCARF Foci p416-Hsp26-EYFP KanMX4, Ura3 pYM39 from EuroSCARF Foci

Table 2: Plasmid-borne fusions of chaperones and fluorescent proteins

Formatted: Centered

Name C-terminal Tag Marker Source of Fluorescent Protein pFA6a-yEmRFP-KanMX6 yEmRFP KanMX6 yEp-GAP-Cherry-GAP-ALG7 (Lorenz) pFA6a-yEmRFP-His5MX6 yEmRFP His5MX6 yEp-GAP-Cherry-GAP-ALG7 (Lorenz) pFA6a-CFP-KanMX6 CFP KanMX6 pFA6a-CFP-His5MX6 CFP His5MX6

Table 3: Constructed plasmids for C-terminal tagging

Formatted: Centered

Name Marker Use p426-Met25c-FFL-GFP Ura3 source eof FFL-GFP p416 Edc3-mCherry, Pab1-CFP Ura3 dual marker plasmid for P-bodies (Edc3) and stress granules (Pab1)

Table 4: Additional plasmids used

Formatted: Centered

Formatted: No widow/orphan control

Formatted: Centered 21

III. Results Formatted: Left, Space After: 8 pt, Line spacing: Multiple 1.08 li, No While investigating chaperone localization and dynamics during regular growth and various widow/orphan control Formatted: stresses, a previous member of our laboratory, Hugo Tapia, Ph.D., observed noticed that Hsp104 , No widow/orphan control when C -terminally fused to GFP, localized to puncta in cells experiencing starvation when C- terminally fused to GFP [93]. A lack of nutrients is not known to cause rapid protein misfolding and aggregation which would attract chaperones, so the presence of Hsp104 in these foci is not as readily explainable as its presence in heat induced foci and t hese puncta looked distinct from heat - induced foci . To compare examine the presence formation of the heat induced foci, known now as q- bodies Q-bodies , and starvation induced foci, which I will call chaperone bodies (CBs), I exposed exponential phase cells from four separate strains cell lines (gHsp104-EGFP, gHsp26-GFP, gHsp42-

GFP, and gSsa1-GF PP) to either a heat shock of 42°C or media lacking glucose for fifteen minutes. I confirmed the formation of q-bodies Q-bodies in live cells using a fluorescence microscope with a filter set appropriate for viewing GFP. Before the heat shock, cells exhibited ha d varying degrees of fluorescence: gHsp104-EGFP had a diffuse localization phenotype , gHsp26-GFP had very little fluorescence, gHsp42-GFP had a punctate localization phenotype , and Ssa1-GFP had a diffuse localization phenotype similar to that of gHsp104-EGFP (Figure 61A). After the heat shock, all cell lines contained a large number of small, overlapping foci (Figure 61B). In gHsp26-GFP and gHsp42-

GFP the foci varied from dim fluorescence to extremely bright, while in gHsp104-EGFP and gSsa1-

GFP the foci were all of similar intensity. Next, I tested for the appearance of CBs in response to a 15 min shift to glucose-free medium. Some cells had fluorescen ce t changes compared to their phenotypes at exponential phase, while others remained the same: gHsp104-EGFP gained one to two puncta in most cells, gHsp26-GFP increased in fluorescence and gained one to two puncta in a fraction of cells, gHsp42-GFP gained a slightly larger fraction of cells with puncta compared to exponential phase of cells with puncta , and Ssa1-GFP remained with a diffuse localization phenotype

Formatted: Centered 22

(Figure 61C). Thus, during exponential phase Hsp42 appears to be localized within c-bodies CBs , while Hsp26 and Hsp104 localize there in response to nutrient deprivation.

To confirm that In order to determine whether glucose-free media doesn’t create s proteotoxic conditions leading that would lead to unfolding of proteins similar to that of heat shock at 42°C, I used a thermolabile model protein, firefly luciferase (FFL), C-terminally fused to G FP F. I o P, and observed whether the protein became unfolded and aggregated during glucose deprivation as it does under heat shock conditions ([63] and Figure 61B). FFL-GFP remained completely diffuse when incubated in glucose-free media (Figure 61C), suggesting that a shift to such media does not result in proteotoxic conditions and instead a unique signal is inducing the chaperones into foci. .

Formatted: Centered 23

Figure 61: Localization of molecular chaperones and firefly luciferase (FFL) in response to heat and starvation. gHsp104-EGFP, gHsp26 -GFP, gHsp42-GFP, gSsa1-GFP and BY4741 + p 426-Met25c-FFL- GFP were grown to exponential phase (A) and then incubated for 15 minutes at 42°C (B) or in the absence of glucose (C).

Formatted: Centered 24

In addition to heat stress and glucose deprivation, cells can encounter many other stresses that could lead to the formation of c-bodies CBs . Accordingly, Thus , I test observ ed the localization of the three chap erones erones that had coalesced into foci during incubation in glucose -free media

(gHsp104-EGFP, gHsp26-GFP, and gHsp42-GFP) during additional stresses including (I) two more energy stresses: 2-deoxy-D-glucose, which is a non-hydrolyzable analog of glucose, and sodium azide, which inhibits cytochrome oxidase B of the electron transport chain; (II) two osmotic stresses:

1M KCl and 1M sorbitol; and (III) depletion of nitrogen, which involved growth in media free of ammonium sulfate. I examined each chaperone and stress in triplicate, capturing images on the fluorescence microscope and quantifying foci per analyzing 100 cells for each sample. Cells expressing gHsp104-GFP display experienc ed a large increase in foci formation between exponential phase and treatment with 2-deoxy-D-glucose and sodium azide, but virtually no increase in foci formation between exponential phase and 1M KCl, 1M sorbitol, or ammonium-sulfate-free media

(Figure 72A). For gHsp26-GFP, the most potent energy stress was sodium azide and even more potent of any stress were the two osmotic stressors (Figure 72B). Like gHsp104-EGFP, incubation of cells expressing Hsp26-GFP did not form foci in response to nitrogen starvation. Hsp42-GFP displayed the same results as Hsp26-GFP except that the increases in foci formation were less robust

(Figure 72C). Thus Hsp104, Hsp26, and Hsp42 all localize to c-bodies CBs during energy stress, the most potent being sodium azide, while both Hsp26 and Hsp42 also coalesce in response to osmotic stress.

Formatted: Centered 25

Formatted: Centered 26

Figure 72: Localization of (A) gHsp104-EGFP, (B) gHsp26-GFP, and (C) gHsp42-GFP in response to Comment [KAM2]: This figure looks blurry heat, energy, osmotic, and nitrogen stresses. White bars represent no treatment, while black bars – I think you need a higher -res version. indicate specified treatment.

Formatted: Centered 27

While heat shock is known to cause aggregation of misfolded proteins and recruitment of

Hsp104, a lack of glucose would not necessarily induce mature proteins in their native state to unfold [64]. However, nutrient deprivation could hamper the folding of newly synthesized proteins by limiting the Hsp70/Hsp40/NEF folding cycle or interfering with ATP-dependent post-translational membrane localization of tail-anchored proteins. To determine whether newly synthesized proteins are necessary components of the foci , I performed the glucose deprivation experiment in the presence of cycloheximide, a potent translation inhibitor. As seen in Figure 83, the presence of cycloheximide completely blocked gHsp104-GFP foci formation in response to glucose deprivation, demonstrating that newly synthesized proteins are required either directly or indirectly for Hsp104 localization to foci. Interestingly, the same did not hold true when cells were exposed to sodium azide: Hsp104-GFP formed foci in the presence of cycloheximide, suggesting a fundamental difference between the foci caused by glucose deprivation and sodium azide treatment, which will be addressed in the discussion.

Several types of foci are known to occur in yeast including p-bodies P-bodies , stress granules, actin bodies, PSGs, and SPGs. In order to determine whether the c-bodie sCBs were components of any of these bodies and, therefore, speculate on their purpose, I created strains in which Hsp104 was fused to one fluorescent protein and a marker representing one of the above mentioned foci was fused to another distinct color . For p-bodies P-bodies and stress granules, I had utilized a dual- marker plasmid containing both Edc3-mCherry and Pab1-CF P P to Hs p104 -EGFP wwhich I transformed into my gHsp104-EGFP strain . To examine possible co-localization of CBs with For actin bodies, PSGs, and SPGs I fused yEmRFP to the C-terminus of the endogenous Hsp104 in three strains: Cap1-GFP, Scl1-GFP, and Hos2-GFP. After a fifteen minute incubation in glucose-free media,

Hsp104 partially colocalizes with proteasome storage granules (Scl1-GFP) and fully overlaps with stationary phase bodies (Hos2-GFP) (Figure 94). Stress granules (Pab1-CFP) did not form foci during Formatted: Centered 28 this acute glucose deprivation, while p-bodies P-bodies (Edc3-mCherry) and actin bodies (Cap1-GFP) formed complexes that were clearly distinct from Hsp104. Thus Hsp104-containing c-bodies CBs may be components of either PSGs or SPGs, the consequences of which will be addressed in the discussion.

Formatted: Centered 29

Figure 83: Localization of Hsp104 -EGFP in response to glucose deprivation (A) and sodium azide (B) in the presence and absence of cycloheximide.

Formatted: Centered 30

Figure 94: Co -expression of Hsp104 -EGFP or Hsp104-yEmRFP with representative markers for various cytoplasmic bodies after 15 minute incubation in glucose -free media.

Formatted: Centered 31

In order to determine whether Hsp26 and Hsp42 were also potential members of PSGs and

SPGs and whether all three chaperones coalesced into one body, I next attempted to construct ed a different ially ly labeled Hsp104 for localization with gHsp26-GFP and gHsp42-GFP. I observed that

Hsp104’s localization patterns were partially dependent on the nature of the fluorescent protein attached. When fused to yEmRFP, Hsp104 remained mostly diffuse during both exponential phase and incubation in glucose free media; this differs from the behavior of gHsp104-GFP which formed

1-2 puncta in most cells in response to starvation (Figure 10). This was unexpected because GFP and yEmRFP, though from different organisms, are similar proteins: both with a molecular weight around 27 kDa arranged in a beta barrel structure. One distinction, however, is that GFP is a weak dimer while yEmRFP has been mutated from an obligate tetramer to a monomer. This distinction leads to the hypothesis that perhaps the mutations in yEmRFP that are causing it to be a monomer are interfering with the aggregation of Hsp104 or that GFP induces the aggregation . Interestingly, gHsp104-yEmRFP had exhibited the same phenotype as gHsp104-EGFP during heat shock at 42°C: many overlapping puncta per cell. This result suggests a fundamental difference between q- bodies Q-bodies and c-bodies CBs since yEmRFP seems to be inhibiting Cc-body B formation but not q- body Q-body formation. Other variants of fluorescent protein displayed additional perturbations in localization pattern. CFP caused Hsp104 to localize to what appeared to be a ring around the bud neck, similar to the septin ring (see [65]), and also to long bands that transversed the entire cell

(Figure 10). Fusion of Hsp104 to CFP also created a cytokinesis defect. In the discussion I will outline several hypotheses rationales for these discrepancies.

Formatted: Centered 32

Figure 10 5: Localization of (A) Hsp104 when fused to different fluorescent proteins. (B) Hsp26 -CFP shows a cytokinesis defect.

Formatted: Centered 33

Formatted: Space Before: 0 pt, No widow/orphan control Comment [KAM3]: Where are your localizations in different knockout experiments? Did none of those work?

Formatted: Centered 34

IV. Discussion Formatted: No widow/orphan control

This thesis explores the subcellular localization of molecular chaperones during exposure of

Saccharomyces cerevisiae to different stresses. One well - studied stress is that of heat shock at 42°C: after a short time at this temperature, chaperones fused to GFP will localize to many, overlapping, cytoplasmic foci. These aggregates were once thought to be haphazard accumulations of proteins, but recent evidence showing that molecular chaperones are required for their appearance and fusion into larger structures, suggests that they may be a regulated aspect of protein quality control. Termed q- bodies Q-bodies , these aggregates associate with the cortical ER membrane, localize with Hsp42 and

Hsp104, and require Hsp82, Ssa1, and Hsp104 to coalesce into larger inclusions. Using the cells lines strains gHsp104-EGFP, gHsp26-GFP, gHsp42-GFP, and gSsa1-GFP, I have confirmed the formation of these chaperones into q-bodies Q-bodies . As mentioned earlier, Dr. Hugo Tapia, Ph.D. a previous member of the laboratory led us to the finding that Hsp104-EGFP assembled into similar complexes when exposed to the stress of glucose deprivation. While not known to cause wide-scale unfolding of proteins,

I have observed this stress to cause causing Hsp104, Hsp26, and Hsp42, but not Ssa1, to assemble into complexes in the cytoplasm. As the heat-induced bodies are hypothesized to be involved in protein refolding or degradation, the starvation-induced bodies may also play a crucial role in protein quality control. The overall goal of my project was to characterize the starvation induced-bodies in terms of composition and dynamics. FMy future directions of this project are to prove that Hsp104, Hsp26, and

Hsp42 are involved in assembly, disassembly, and/or recruitment of other constituents, and that focus formation is a cytoprotective response to maintain protein homeostasis.

The three chaperones that displayed starvation-induced foci, Hsp104, Hsp26, and Hsp42, had distinct behavior phenotype s during both exponential phase and resuspension in glucose-free media. gHsp104-EGFP went from mostly diffuse to 1-2 puncta in most cells, Hsp26 went from unexpressed to puncta in a fraction of cells, while Hsp42 coalesced into puncta during both treatments. These data Formatted: Centered 35 suggest that Hsp42 may be poised in the cytoplasm waiting for Hsp104 and Hsp26 to be specifically recruited or induced upon nutrient starvation. For example, in SPGs, Hsp42 wa is needed for the bodies to form: it was a founding member that had to arrive before others could be recruited [52]. Hsp42- containing c-bodies CBs may be ubiquitous sites of protein quality control that enlist the help of Hsp26 and Hsp104 in times of starvation.

Since Hsp26, Hsp42, and Hsp104 bind to misfolded proteins and aggregates, I wanted to determine whether glucose deprivation is somehow leading to the unfolding of proteins. By fusing GFP to the thermally-labile FFL, I observed that this protein does was not , in fact, aggregat eing in response to growth in glucose-free media, suggesting that the qualities that make this protein unfold in the presence of heat are not present due to starvation. This does not preclude the possibility that a different subset of proteins is unfolding in this situation. For example, energy, in the form of ATP, is required for the folding of newly synthesized proteins via Hsp70/Hsp40/NEF and Hsp90 as well as for the localization of tail- anchored membrane proteins via Get3, an ATPase responsible for shuttling these proteins through the cytoplasm. To determine whether newly synthesized proteins were coalescing with the chaperones, I performed the glucose deprivation experiment in the presence of cycloheximide, an inhibitor of the ribosome. I found that cycloheximide completely inhibited the formation of Hsp104 foci, meaning that protein synthesis must be actively occurring for the bodies to form. Strangely, the same experiment with a sodium azide stress instead of glucose deprivation did not yield the same result: Hsp104 was able to assemble into complexes despite the absence of protein synthesis. This implies that glucose deprivation and sodium azide are not the same type of stress. During exponential phase, Saccharomyces cerevisiae will ferment all glucose into ethanol before switching to aerobic respiration on the ethanol. Thus, glucose deprivation will cause the cell to cease growing almost immediately because it will remove all glucose without a buildup of ethanol .; Conversely on the other hand , sodium azide, which, as mentioned above, inhibits cyctochrome oxidase B, only affects aerobic respiration and will allow the cell to continue Formatted: Centered 36 fermenting and growing until all the glucose is used up. Since sodium azide is seen to cause chaperone puncta as quickly as glucose deprivation, perhaps the stress is not directly energy related. Indeed, sodium azide is known to increase thermotolerance which would imply ing induction of the heat shock response that it is able to turn on the heat shock response . To investigate this anomaly, I could perform the cycloheximide experiment with simultaneous glucose deprivation and sodium azide treatment: if the sodium azide is its own separate stress, then puncta should form despite the lack of protein synthesis.

Saccharomyces cerevisiae and other eukaryotic cells form distinct and regulated cytoplasmic multi-protein complexes in response to various physiological stresses. Examples of these structures include p-bodies P-bodies , stress granules, actin bodies, PSGs, and SPGs. By using a fluorescent protein fused to the C-terminus of a marker protein from each of the mentioned complexes, I tested for colocalization with gHsp104-EGFP or gHsp104-yEmRFP after fifteen minutes incubation in glucose-free media. Both p-bodies P-bodies and stress granules have been shown to appear after acute glucose deprivation [49]. Edc3, a decapping activator that localizes to p-bodies P-bodies , fused to mCherry formed discrete puncta after fifteen minutes but the majority of foci did not overlap with gHsp104-EGFP foci, suggesting that Hsp104 does not localize to these sites of concentrated mRNA degradation machinery. Interestingly, I (and others from our laboratory (Dr. Kevin Morano, Ph.D., personal communication) ) could not get stress granules, as visualized by the poly(A) binding protein, Pab1, fused to CFP, to appear during an acute glucose deprivation; thus, the Hsp104 granules do not coalesce with the complexes of translation initiation proteins during 15 min starvation.

I tested additional complexes, known for their appearance during stationary phase, for formation after acute glucose deprivation. Quiescence, or stationary phase, is a set of cellular conditions activated in response to extended growth in nutrient-free media. It is possible, as shown above below , that some of these bodies would begin to form shortly after glucose is removed from the media in preparation for a transition into stationary phase. When cells are in a quiescent state, cell growth and Formatted: Centered 37 division as well as transcription and translation are halted, chromosomes are condensed, mRNA degradation is repressed, the cell wall is strengthened, and the cell becomes more resistant to stresses.

If the cell is not growing, actin is not needed and is, thus, stored with actin binding proteins in actin bodies. Cap1, responsible for capping the ends of actin filaments, frequently formed puncta when fused to GFP at a great frequency , but they were completely distinct from the localization of gHsp104-EGFP.

Proteasome storage granules, on the other hand, represented by Scl1, a subunit of the proteasome, fused to GFP overlapped partially with Hsp104-yEmRFP. Scl1 appeared to be diffuse throughout the nucleus as well as in juxtanuclear puncta that colocalized with Hsp104 puncta. Throughout most of the cell cycle, the majority of 26 Ss proteasomes are localized to the nucleus. They are responsible for degrading many cell cycle regulators and, thus, are essential for cell cycle progression. During entry into stationary phase, proteasome subunits relocalize to cytoplasmic foci for storage. Perhaps Hsp104 is aiding in the assembly of these complexes. Important future experiments would be to observe formation of proteasome storage granules in cells where Hsp104 has been genetically ablated, examining whether the bodies formed at all and the speed in which they did, and whether the cells had a slower transition from exponential to stationary or from stationary back to exponential.

In addition to the PSGs, Hsp104 also colocalized with Hos2, a nuclear histone deacetylase that is shuttled into cytoplasmic bodies termed stationary phase granules during the transition from exponential phase to quiescence. Cells lacking Hos2 are more likely than wild-type cells to die during stationary phase and the cells that do survive take significantly longer to recover to exponential growth, suggesting that Hos2 is a major player in stationary phase survival and that the SPGs serve an important function [42]. The fact that Hos2 is upregulated during stationary phase lends further credibility to this hypothesis [48, 52]. Additional proteins found in the bodies include Hst2 (a cytoplasmic protein deacetylase), Set3 (a histone deacetylase involved in meiotic gene repression), Yca1 (a cysteine protease involved in cell cycle dynamics), Hsp26, Hsp42, and some stress granule components. Hsp42 is essential Formatted: Centered 38 for formation of the complexes. Perhaps Hsp104 is also responsible for the assembly of these bodies either separately or in conjunction with the PSGs. Experiments with Hsp104-yEmRFP, Scl1-GFP, and

Hos2-CFP, for example, would reveal whether the three are going to one complex or whether they are distinct bodies. Similarly, experiments where Hsp104 was deleted from the genome would exemplify tell the importance of the chaperone in assembling the bodies. Interestingly, Hst2 was not found in the complexes after acute glucose deprivation: it only appeared after cells reached stationary phase of growth. This suggests that Hsp104 and Hos2 might be early members of the bodies, while Hst2 joins them further along in time. To analyze kinetics of foci formation, I can perform future experiments can be performed in a microfluidic chamber made by CellASIC that can be viewed on a fluorescence microscope. Cells are trapped in a single focal plane by an elastic membrane and the experimenter can dictate the type and duration of media that flows through the chamber. TI can supply t he system can be supplied with glucose-free media and observe for each cell the timing and formation of Hos2-GFP,

Hsp104-yEmRFP, and Hst2-CFP granules.

The fact that SPGs colocalize with Hsp104 and also were shown to contain Hsp26 and Hsp42 provide indirect evidence that Hsp104, Hsp26, and Hsp42 are coalescing into the same bodies. For more concrete proof, I attempted to fuse different fluorescent proteins to the C-terminus of Hsp104 for colocalization studies with Hsp26-GFP and Hsp42-GFP. I discovered that these fluorescent proteins partially affect the localization of Hsp104 in starvation induced foci. I began by fusing yEmRFP to the C- terminus of endogenous Hsp104. I found no increase between the number of foci present during exponential phase and the number present during glucose deprivation. The protein yEmRFP is derived from the coral Discosoma , while GFP is derived from the jellyfish Aequoreia victoria ; however, they are nearly identical in molecular weight and have similar beta barrel structures. The main difference is that yEmRFP was mutated to a monomer from an obligate tetramer, while GFP remains a weak dimer.

Perhaps some of these mutations affect Hsp104’s ability to oligomerize. By fusing the protein to the C- Formatted: Centered 39 terminus of Edc3, the p-body P-body marker used above, and observing the formation of p-bodies P- bodies , we could determine whether this problem was specific to Hsp104 or similar for other starvation induced bodies.

Strangely, when Hsp104 was fused to CFP, a third phenotype occurred: Hsp104 localized to what appeared to be a ring around the bud neck and long bands across the cell. Additionally there was a cytokinesis defect where cells would grow into elongated forms. CFP is a mutant form of GFP with a

66 66 single amino acid substitution (Try to Trp ) and is thus extremely similar to GFP. Formatted: Superscript Formatted: Superscript The ring-like structure looks similar to the septin ring and the bands resemble Gln1 filaments that form in response to starvation [66]. This phenotype may actually provide important information on

Hsp104’s activities in the cell: perhaps it does not normally aggregate at the septin ring and Gln1 filaments, but when attached to CFP its kinetics are slowed down and we can observe it stalled at these places. Hsp26 and Hsp42 when fused to CFP provided the same phenotype suggesting that all three chaperones are involved in the assembly or disassembly of these rings and filaments. These data open up a whole new pathway of investigation of the chaperones’ subcellular localization. The first steps on this path would be to perform colocalization experiments with fluorescently labelled septin and Gln1, respectively. Due to these discrepancies in fluorescent protein localization, the next step forward in this project would be to confirm the localization via indirect immunofluorescence using an antibody against each wild-type chaperone to eliminate the potential of the fluorescent moiety influencing the behavior of the chaperone.

In the following paragraphs I will outline future lines of experimentation for this project. These include finding additional proteins (if any) that localize to the CBs, determining which components are required for assembly and disassembly, examining whether the disaggregase activity of Hsp104 is essential for normal CB CB dynamics, and analyzing the contribution of CBs to fitness and survival of Formatted: Centered 40 stress.

To determine all of the additional proteins that coalesce with Hsp26, Hsp42, and Hsp104 in response to removal of glucose from the media, I could attempt to immunoprecipitat e aggregates of each of the chaperones from glucose deprived cells could be immunoprecipitated and identify the proteins that co-precipitate could be identified via mass spectrometry. I anticipate that a small number of proteins (around 10, based on the number of proteins believed to be in the other stress-induced cytoplasmic assemblies described in the literature) will co-precipitate with the chaperones. TI can confirm t he presence of these proteins in c-bodies CBs can be confirmed by translationally fusing yEmRFP to the C-termini of each of the candidate proteins and observing their localization in comparison to Hsp26-GFP, Hsp42-GFP, and Hsp104-EGFP during glucose deprivation.

Certain constituents of chaperone bodies may be required for their assembly and/or disassembly. Since Hsp104 is the main disaggregase in the yeast cell, it may be required specifically for disassembly of the complexes. In P-bodies, for example, the mRNA decapping factor Pat1 is required as a scaffold upon which the other constituents assemble [60]. Phosphorylation of Pat1 by protein kinase A

(PKA) results in disassembly of the structures [60]. Each chaperone’s necessity could be determined by genetic ablation and observation of whether the bodies still form and with the same kinetics and frequency via a second fluorescently fused constituent. For example, in order to determine the necessity of Hsp104 for assembly, the number of Hsp26-YFP (expressed from a plasmid: see Table 2) foci can be compared between a wild type strain and a strain in which Hsp104 has been genetically ablated— hsp104Δ. If Hsp104 is important for assembly of Hsp26 then there should be fewer Hsp26 foci in the deletion mutant. This experiment can be repeated with an Hsp42-YFP plasmid to determine the necessity of Hsp104 in Hsp42 assembly. Future experiments in the laboratory involving hsp26Δ, hsp42Δ, and p416-Hsp104-YFP will need to be undertaken to delineate the necessity of Hsp26 and Hsp42 in assembly of CBs. Additionally the dissolution of CBs in the deletion mutants will uncover whether any of Formatted: Centered 41 the chaperones are involved in disassembly of the aggregates.

Certain constituents of chaperone bodies may be required for their ass embly and/or disassembly. Since Hsp104 is the main disaggregase in the yeast cell, it may be required specifically for disassembly of the complexes. In p-bodies , for example, the mRNA decapping factor Pat1 is required as a scaffold upon which the other constituents assemble [60]. Phosphorylation of Pat1 by protein kinase A

(PKA) results in disassembly of the structures [60]. For e ach chaperone , I could determine its necessity by genetic ablation and observation of whether the bodies still form and with the same kinetics and frequen cy via a second fluorescently fused constituent. I have preliminary evidence that none of the three chaperones are required for the others to form bodies. Comment [KC4]: More detail here or promote to results section? In order to determine whether the disaggregase activity of Hsp104 is essential for regular CB CB dynamics, I can use several disaggregase-deficient mutant versions of Hsp104 can be used . Hsp104 Y662A contains a mutation in the tyrosine residue of the axial channel loop of nucleotide binding domain 2

(NBD2) that is required for substrate threading and can neither resolubilize aggregated proteins nor provide thermotolerance to the cell [61]. Hsp104 E645K contains a mutation in the axial channel of NBD2, which results in impaired movement of the tyrosine loop and the same phenotype as the non- conservative mutation in the loop itself [61]. Hsp104 E285Q/E687Q contains one mutation in each of the

ATPase domains resulting in a protein that irreversibly binds substrates and, thus, can neither resolubilize them nor provide thermotolerance [62]. I anticipate that the disaggregase activity of Hsp104 is required for efficient disassembly of the CBs. If this prediction is true, then cells containing the mutant

Hsp104s will have more Hsp42-GFP and Hsp26-GFP foci remaining per minute after recovery in SC media or will retain a total overall number of Hsp26-GFP and Hsp42-GFP foci at the end of a fifteen minute recovery period.

Additionally, I can use a a high-throughput spotted microarray technique developed by

Narayanaswamy et al. can be used to uncover any proteins that may have an effect on assembly and Formatted: Centered 42 disassembly [63]. The technique involves transforming a deletion library with a plasmid containing

Hsp104-EGFP, exposing the cells to either rich media or media lacking glucose, and “printing” the cells from a 96-well plate onto a polylysine-coated, glass microscope slide using a robotic apparatus. If a protein is a positive regulator of CBs (either by promoting assembly or inhibiting disassembly), then I expect that when it is absent, cells will have fewer Hsp104-EGFP and Hsp42-GFP foci during glucose deprivation. On the other hand, if a protein is a negative regulator of CBs (either by inhibiting assembly or promoting disassembly), then I expect that when it is absent , cells will have more Hsp104-GFP and

Hsp42-GFP foci when cells are growing in glucose-replete media.

DI would also like to d etermin ing e the contributions of CBs to fitness, growth, and long-term survival of the cell is also important . The formation of p-bodies P-bodies , for example, has been strongly correlated to increased survival during stationary phase. Pat1, an mRNA decapping factor, inhibits P- body P-body formation when phosphorylated on two particular serine residues [60]. When these serine residues are substituted with aspartic acid residues the resulting protein (Pat1-EE) behaves as if it is constitutively phosphorylated and produces few p-bodies P-bodies in response to entry into stationary phase [64]. Cells containing Pat1-EE exhibit reduced survival after ten days of growth [64]. The authors concluded that the reduction in P-body P-body formation rather than a secondary effect of mutating

Pat1 was responsible for the loss of viability due to two facts: protein levels of Pat1-EE were similar to those of wild-type Pat1 and the overexpression of Pat1-EE resulted in the same phenotype (growth arrest due to increased translational repression) as the overexpression of wild-type Pat1 [64]. Since CBs form under a similar stress to p-bodies P-bodies and have a similar morphology, I hypothesize that CBs also have cytoprotective effects during glucose deprivation.

For my first line of experimentation First , a rebudding assay could be performed I would perform a rebudding assay where I compare t the time elapsed between addition of glucose to cells undergoing glucose deprivation to the resumption of cell division is compared in CB-positive and CB-negative cells in Formatted: Centered 43 order to determine whether CBs are essential for efficient return to regular cellular activities My hypothesis predicts that the length of time elapsed between the replenishment of glucose to the media and the resumption of budding will be longer in CB-negative cells, signifying that CBs are important for the quick and efficient resumption of regular cellular activities perhaps by sequestering constituents from degradation to prevent the need for resynthesis upon removal of the stress or by concentrating constituents for streamlined functions (specified by whichever proteins are contained within CBs). On the other hand, it is possible that the length of time before rebudding will be shorter in CB-negative cells, signifying that CBs may be sequestering proteins involved in growth and cell division presumably hindering the cells from dividing when conditions are not favorable.

UI can use uptake of propidium iodide, a fluorescent molecule to which live cells are impenetrable, can be used as an assay to distinguish between live and dead cells in order to determine whether CBs promote survival during glucose deprivation. My prediction that CBs are important for survival in nutrient sparse media will be supported if significantly more CB-negative cells die.

One hypothesis for the function of the CBs is that they serve to protect their constituent proteins from degradation during times of stress. To test this hypothesis, I can perform a degradation assay involving Western blots can be performed . By comparing the amount of protein (as measured by the intensity of the band) between wild-type cells and those deficient for CBs, I can det ermine which proteins that are protected from degradation by the presence of the complexes can be detected . If CBs serve to protect a particular constituent during glucose deprivation, then I would expect the CB-positive experimental group to have an equally intense protein band (measured as fluorescence intensity from the secondary antibody) for each of the time points and the CB-negative experimental group to have progressively weaker bands for each time point from T0 onward. I would expect there to be no differences between the CB-positive and CB-negative control groups that were not deprived of glucose.

In conclusion, I have shown that Hsp104 coalesces into cytoplasmic complexes in response to Formatted: Centered 44 nutrient stresses while Hsp26 and Hsp42 exhibit the same behavior for both nutrient and osmotic stresses. I have shown that nutrient stress does not cause large-scale unfolding of proteins as heat stress does for thermolabile proteins. Hsp104 bodies formed due to glucose deprivation require newly synthesized proteins while similar bodies formed due to exposure to sodium azide do not. Hsp104 colocalizes with members of both proteasome storage granules and stationary phase granules. I have outlined some future lines of experimentation that will hopefully uncover the fitness benefits of these chaperone bodies and possibl ye shed light on other known stress-induced complexes.

Nice writing in the discussion – change to not first person and it’s all set.

Formatted: Centered 45

Formatted: Centered 46

References

1. Fink, A.L., Chaperone-mediated protein folding. Physiol. Rev., 1999. 79 (2): p. 425-449. 2. Verghese, J., J. Abrams, Y. Wang, and K.A. Morano, Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol. Mol. Biol. Rev., 2012. 76 (2): p. 115-158. 3. Hartl, F.U., Molecular chaperones in cellular protein folding. 1996. 4. Horwich, A.L., Molecular Chaperones in Cellular Protein Folding: The Birth of a Field. Cell, 2014. 157 (2): p. 285-288. 5. Herczenik, E. and M.F. Gebbink, Molecular and cellular aspects of protein misfolding and disease. FASEB J., 2008. 22 (7): p. 2115-2133. 6. Escusa-Toret, S., W.I. Vonk, and J. Frydman, Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nat. Cell Biol., 2013. 15 (10): p. 1231-1243. 7. Williams, D.C., R.M. Van Frank, W.L. Muth, and J.P. Burnett, Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science, 1982. 215 (4533): p. 687- 689. 8. de Groot, N.S., R. Sabate, and S. Ventura, Amyloids in bacterial inclusion bodies. Trends Biochem. Sci., 2009. 34 (8): p. 408-416. 9. Serio, T.R. and S.L. Lindquist, Protein-only inheritance in yeast: something to get [PSI+]-ched about. Trends Cell Biol., 2000. 10 (3): p. 98-105. 10. Halfmann, R., D.F. Jarosz, S.K. Jones, A. Chang, A.K. Lancaster, and S. Lindquist, Prions are a common mechanism for phenotypic inheritance in wild yeasts. Nature, 2012. 482 (7385): p. 363- 368. 11. Vashist, S., M. Cushman, and J. Shorter, Applying Hsp104 to protein-misfolding disorders. Biochem. Cell Biol., 2010. 88 (1): p. 1-13. 12. Doyle, S.M. and S. Wickner, Hsp104 and ClpB: protein disaggregating machines. Trends Biochem. Sci., 2009. 34 (1): p. 40-48. 13. Shorter, J., Hsp104: a weapon to combat diverse neurodegenerative disorders. Neurosignals, 2007. 16 (1): p. 63-74. 14. Weibezahn, J., C. Schlieker, P. Tessarz, A. Mogk, and B. Bukau, Novel insights into the mechanism of chaperone-assisted protein disaggregation. Biol. Chem., 2005. 386 (8): p. 739-744. 15. Sanchez, Y., J. Taulien, K. Borkovich, and S. Lindquist, Hsp104 is required for tolerance to many forms of stress. EMBO J., 1992. 11 (6): p. 2357. 16. Sanchez, Y. and S.L. Lindquist, HSP104 required for induced thermotolerance. Science, 1990. 248 (4959): p. 1112-1115. 17. Tessarz, P., M. Schwarz, A. Mogk, and B. Bukau, The yeast AAA+ chaperone Hsp104 is part of a network that links the actin cytoskeleton with the inheritance of damaged proteins. Mol. Cell. Biol., 2009. 29 (13): p. 3738-3745. 18. Chernoff, Y.O., S.L. Lindquist, B.-i. Ono, S.G. Inge-Vechtomov, and S.W. Liebman, Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science, 1995. 268 (5212): p. 880-884. 19. Wendler, P., J. Shorter, C. Plisson, A.G. Cashikar, S. Lindquist, and H.R. Saibil, Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. Cell, 2007. 131 (7): p. 1366-1377. 20. Wendler, P., J. Shorter, D. Snead, C. Plisson, D.K. Clare, S. Lindquist, and H.R. Saibil, Motor mechanism for protein threading through Hsp104. Mol. Cell, 2009. 34 (1): p. 81-92.

Formatted: Centered 47

21. Parsell, D.A., A.S. Kowal, and S. Lindquist, Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes. J. Biol. Chem., 1994. 269 (6): p. 4480- 4487. 22. Schirmer, E.C., C. Queitsch, A.S. Kowal, D.A. Parsell, and S. Lindquist, The ATPase activity of Hsp104, effects of environmental conditions and mutations. J. Biol. Chem., 1998. 273 (25): p. 15546-15552. 23. Bösl, B., V. Grimminger, and S. Walter, Substrate binding to the molecular chaperone Hsp104 and its regulation by nucleotides. J. Biol. Chem., 2005. 280 (46): p. 38170-38176. 24. Schaupp, A., M. Marcinowski, V. Grimminger, B. Bösl, and S. Walter, Processing of proteins by the molecular chaperone Hsp104. J. Mol. Biol., 2007. 370 (4): p. 674-686. 25. Winkler, J., J. Tyedmers, B. Bukau, and A. Mogk, Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation. J. Cell Biol., 2012. 198 (3): p. 387-404. 26. Doyle, S.M., J.R. Hoskins, and S. Wickner, Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system. Proc. Natl. Acad. Sci. USA, 2007. 104 (27): p. 11138- 11144. 27. Doyle, S.M., J. Shorter, M. Zolkiewski, J.R. Hoskins, S. Lindquist, and S. Wickner, Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat. Struct. Mol. Biol., 2007. 14 (2): p. 114-122. 28. Lee, S., B. Sielaff, J. Lee, and F.T. Tsai, CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation. Proc. Natl. Acad. Sci. USA, 2010. 107 (18): p. 8135-8140. 29. Haslbeck, M., N. Braun, T. Stromer, B. Richter, N. Model, S. Weinkauf, and J. Buchner, Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae. EMBO J., 2004. 23 (3): p. 638-649. 30. Caughey, B. and P.T. Lansbury Jr, PROTOFIBRILS, PORES, FIBRILS, AND NEURODEGENERATION: Separating the Responsible Protein Aggregates from The Innocent Bystanders*. Annu. Rev. Neurosci., 2003. 26 (1): p. 267-298. 31. Kholova, I. and H. Niessen, Amyloid in the cardiovascular system: a review. J. Clin. Pathol., 2005. 58 (2): p. 125-133. 32. Skovronsky, D.M., V.M.Y. Lee, and J.Q. Trojanowski, Neurodegenerative diseases: new concepts of pathogenesis and their therapeutic implications. Annu. Rev. Pathol. Mech. Dis., 2006. 1: p. 151-170. 33. Taylor, J.P., J. Hardy, and K.H. Fischbeck, Toxic proteins in neurodegenerative disease. Science, 2002. 296 (5575): p. 1991-1995. 34. Baxa, U., V. Speransky, A.C. Steven, and R.B. Wickner, Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein. Proc. Natl. Acad. Sci. USA, 2002. 99 (8): p. 5253-5260. 35. Dobson, C.M., Protein folding and misfolding. Nature, 2003. 426 (6968): p. 884-890. 36. Eisenberg, D., R. Nelson, M.R. Sawaya, M. Balbirnie, S. Sambashivan, M.I. Ivanova, A.Ø. Madsen, and C. Riekel, The structural biology of protein aggregation diseases: Fundamental questions and some answers. Accounts of chemical research, 2006. 39 (9): p. 568-575. 37. Knowles, T.P., A.W. Fitzpatrick, S. Meehan, H.R. Mott, M. Vendruscolo, C.M. Dobson, and M.E. Welland, Role of intermolecular forces in defining material properties of protein nanofibrils. Science, 2007. 318 (5858): p. 1900-1903. 38. Smith, J.F., T.P.J. Knowles, C.M. Dobson, C.E. MacPhee, and M.E. Welland, Characterization of the nanoscale properties of individual amyloid fibrils. Proc. Natl. Acad. Sci. USA, 2006. 103 (43): p. 15806-15811. 39. Shaner, L., H. Wegele, J. Buchner, and K.A. Morano, The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb. J. Biol. Chem., 2005. 280 (50): p. 41262-41269. Formatted: Centered 48

40. Rampelt, H., J. Kirstein-Miles, N.B. Nillegoda, K. Chi, S.R. Scholz, R.I. Morimoto, and B. Bukau, Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO J., 2012. 41. Wenk, G.L., Neuropathologic changes in Alzheimer's disease. J. Clin. Psychiatr., 2003. 64 : p. 7-10. 42. Arimon, M., V. Grimminger, F. Sanz, and H.A. Lashuel, Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Aβ fibrils and protofibrils. J. Mol. Biol., 2008. 384 (5): p. 1157-1173. 43. Braak, H., K.D. Tredici, U. Rüb, R.A.I. de Vos, E.N.H. Jansen Steur, and E. Braak, Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging, 2003. 24 (2): p. 197-211. 44. Bianco, C.L., J. Shorter, E. Régulier, H. Lashuel, T. Iwatsubo, S. Lindquist, and P. Aebischer, Hsp104 antagonizes α-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease. J. Clin. Invest., 2008. 118 (9): p. 3087. 45. Vacher, C., L. Garcia-Oroz, and D.C. Rubinsztein, Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington's disease. Hum. Mol. Genet., 2005. 14 (22): p. 3425-3433. 46. Dandoy-Dron, F., A. Bogdanova, V. Beringue, Y. Bailly, M.G. Tovey, H. Laude, and M. Dron, Infection by ME7 prion is not modified in transgenic mice expressing the yeast chaperone Hsp104 in neurons. Neurosci. Lett., 2006. 405 (3): p. 181-185. 47. Mosser, D.D., S. Ho, and J.R. Glover, Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling. Biochemistry, 2004. 43 (25): p. 8107-8115. 48. Glover, J.R. and S. Lindquist, Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell, 1998. 94 (1): p. 73. 49. Balagopal, V. and R. Parker, Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr. Opin. Cell Biol., 2009. 21 (3): p. 403-408. 50. Sagot, I., B. Pinson, B. Salin, and B. Daignan-Fornier, Actin bodies in yeast quiescent cells: an immediately available actin reserve? Mol Biol Cell, 2006. 17 (11): p. 4645-55. 51. Laporte, D., B. Salin, B. Daignan-Fornier, and I. Sagot, Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J. Cell Biol., 2008. 181 (5): p. 737-45. 52. Liu, I.C., S.W. Chiu, H.Y. Lee, and J.Y. Leu, The histone deacetylase Hos2 forms an Hsp42- dependent cytoplasmic granule in quiescent yeast cells. Mol Biol Cell, 2012. 23 (7): p. 1231-1242. 53. Narayanaswamy, R., M. Levy, M. Tsechansky, G.M. Stovall, J.D. O'Connell, J. Mirrielees, A.D. Ellington, and E.M. Marcotte, Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation. Proc. Natl. Acad. Sci. USA, 2009. 106 (25): p. 10147-10152. 54. Cebollero, E. and F. Reggiori, Regulation of autophagy in yeast Saccharomyces cerevisiae. BBA Molecular Cell Research, 2009. 1793 (9): p. 1413-1421. 55. Laporte, D., B. Salin, B. Daignan-Fornier, and I. Sagot, Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J. Cell Biol., 2008. 181 (5): p. 737-745. 56. Klingauf, M., D. Stanĕk, and K.M. Neugebauer, Enhancement of U4/U6 small nuclear ribonucleoprotein particle association in Cajal bodies predicted by mathematical modeling. Mol Biol Cell, 2006. 17 (12): p. 4972-4981. 57. Arimoto, K., H. Fukuda, S. Imajoh-Ohmi, H. Saito, and M. Takekawa, Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nat. Cell Biol., 2008. 10 (11): p. 1324-1332. 58. Coller, J. and R. Parker, Eukaryotic mRNA decapping. Annu. Rev. Biochem., 2004. 73 (1): p. 861- 890. 59. DeRisi, J.L., V.R. Iyer, and P.O. Brown, Exploring the metabolic and genetic control of gene expression on a genomic scale. Science, 1997. 278 (5338): p. 680-686. Formatted: Centered 49

60. Davie, J.K., D.G. Edmondson, C.B. Coco, and S.Y.R. Dent, Tup1-Ssn6 interacts with multiple class I histone deacetylases in vivo. J. Biol. Chem., 2003. 278 (50): p. 50158-50162. 61. Longtine, M.S., A. McKenzie III, D.J. Demarini, N.G. Shah, A. Wach, A. Brachat, P. Philippsen, and J.R. Pringle, Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 1998. 14 (10): p. 953-961. 62. Swisher, K.D. and R. Parker, Localization to, and effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on stress granules in Saccharomyces cerevisiae. PloS one, 2010. 5(4): p. e10006. 63. Frydman, J., H. Erdjument-Bromage, P. Tempst, and F.U. Hartl, Co-translational domain folding as the structural basis for the rapid de novo folding of firefly luciferase. Nat. Struct. Mol. Biol., 1999. 6(7): p. 697-705. 64. Specht, S., S.B. Miller, A. Mogk, and B. Bukau, Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae. J. Cell Biol., 2011. 195 (4): p. 617- 629. 65. Longtine, M.S., C.L. Theesfeld, J.N. McMillan, E. Weaver, J.R. Pringle, and D.J. Lew, Septin- dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae. Mol. Cell. Biol., 2000. 20 (11): p. 4049-4061. 66. Petrovska, I., E. Nüske, M.C. Munder, G. Kulasegaran, L. Malinovska, S. Kroschwald, D. Richter, K. Fahmy, K. Gibson, and J.-M. Verbavatz, Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation. eLife, 2014. 3.

Formatted: Line spacing: Double, No widow/orphan control

Formatted: No widow/orphan control

Formatted: Centered 50

Kimberly Jaclyn Cope was born in Walnut Creek, CA on September 8 th , 1984. She is the daughter of Isobel ( Mcleod McLeod ) Cope and Ronald Samuel Cope. After graduating from Las Lomas High School in 2002, she attended the University of California, Davis. She graduated with honors with a Bachelor of Sciences in Biology with an emphasis in Molecular and Cellular Biology. In 2009, she entered The University of Texas Graduate School of Biomedical Sciences at Houston.

Permanent address: 2136 Wilmington Dr Walnut Creek, CA 94596

Formatted: Centered 51